ON-TARGETplus Human STAT6 (6778) siRNA - SMARTpool

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	Normal adult human keratinocytes were obtained by trypsinization of skin samples from patients undergoing plastic surgery as previously described [13]. Second-passage keratinocytes were grown in keratinocyte serum-free medium (KSFM) (Gibco, Invitrogen, Carlsbad, CA). 24 hours before stimulation with IFNα (1000 U/ml, cat. no. 11200–1, R&D Systems, Oxon, UK), the medium was changed to keratinocyte basal medium (KBM, the same as KSFM but without growth factors) in which the cells were stimulated. Cells were grown at 37˚C and 5% CO2 in an incubator. The Regional Ethical Committee of Region Midtjylland, Denmark approved the experiments with cultured human keratinocytes (M-20110027). Cultured human keratinocytes were grown to 60–70% confluency. Before transfection, the cells were changed to medium without growth factors (KBM). siRNA directed against STAT6 (cat. no. L-006690-00-0005; Dharmacon, Lafayette, CO) was preincubated with Dharmafect-2 transfection reagent (Dharmacon) in KBM for 20 minutes. The formed siRNA/transfection reagent complexes were added to the cells to a final concentration of 10 nM. As negative controls, cells were transfected with siControl nontargeting pool siRNA (cat. no. D001810-10-05, Dharmacon) or the transfection reagent alone (mock). Five hours after transfection, the medium was changed to keratinocyte growth medium (growth factors included). 24 hours before stimulation, the medium was changed to KBM

Protocol tips

Normal adult human keratinocytes were obtained by trypsinization of skin samples from patients undergoing plastic surgery as previously described [13]. Second-passage keratinocytes were grown in keratinocyte serum-free medium (KSFM) (Gibco, Invitrogen, Carlsbad, CA). 24 hours before stimulation with IFNα (1000 U/ml, cat. no. 11200–1, R&D Systems, Oxon, UK), the medium was changed to keratinocyte basal medium (KBM, the same as KSFM but without growth factors) in which the cells were stimulated. Cells were grown at 37˚C and 5% CO2 in an incubator. The Regional Ethical Committee of Region Midtjylland, Denmark approved the experiments with cultured human keratinocytes (M-20110027). Cultured human keratinocytes were grown to 60–70% confluency. Before transfection, the cells were changed to medium without growth factors (KBM). siRNA directed against STAT6 (cat. no. L-006690-00-0005; Dharmacon, Lafayette, CO) was preincubated with Dharmafect-2 transfection reagent (Dharmacon) in KBM for 20 minutes. The formed siRNA/transfection reagent complexes were added to the cells to a final concentration of 10 nM. As negative controls, cells were transfected with siControl nontargeting pool siRNA (cat. no. D001810-10-05, Dharmacon) or the transfection reagent alone (mock). Five hours after transfection, the medium was changed to keratinocyte growth medium (growth factors included). 24 hours before stimulation, the medium was changed to KBM

Publication protocol

Normal adult human keratinocytes were obtained by trypsinization of skin samples from patients undergoing plastic surgery as previously described [13]. Second-passage keratinocytes were grown in keratinocyte serum-free medium (KSFM) (Gibco, Invitrogen, Carlsbad, CA). 24 hours before stimulation with IFNα (1000 U/ml, cat. no. 11200–1, R&D Systems, Oxon, UK), the medium was changed to keratinocyte basal medium (KBM, the same as KSFM but without growth factors) in which the cells were stimulated. Cells were grown at 37˚C and 5% CO2 in an incubator. The Regional Ethical Committee of Region Midtjylland, Denmark approved the experiments with cultured human keratinocytes (M-20110027). Cultured human keratinocytes were grown to 60–70% confluency. Before transfection, the cells were changed to medium without growth factors (KBM). siRNA directed against STAT6 (cat. no. L-006690-00-0005; Dharmacon, Lafayette, CO) was preincubated with Dharmafect-2 transfection reagent (Dharmacon) in KBM for 20 minutes. The formed siRNA/transfection reagent complexes were added to the cells to a final concentration of 10 nM. As negative controls, cells were transfected with siControl nontargeting pool siRNA (cat. no. D001810-10-05, Dharmacon) or the transfection reagent alone (mock). Five hours after transfection, the medium was changed to keratinocyte growth medium (growth factors included). 24 hours before stimulation, the medium was changed to KBM

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